Ceruloplasmin is an endogenous inhibitor of myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nM

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules

Hypochlorous acid inactivates myeloperoxidase inside phagocytosing neutrophils

When neutrophils phagocytose bacteria, they release myeloperoxidase (MPO) into phagosomes to catalyse the conversion of superoxide to the potent antimicrobial oxidant hypochlorous acid (HOCl). Here we show that within neutrophils, MPO is inactivated by HOCl. In this study, we aimed to identify the effects of HOCl on the structure and function of MPO, and determine the enzyme’s susceptibility to oxidative inactivation during phagocytosis. When hydrogen peroxide was added to a neutrophil granule extract containing chloride, MPO activity was rapidly lost in a HOCl-dependent reaction. With high concentrations of hydrogen peroxide, western blotting demonstrated that MPO was both fragmented and converted to high molecular weight aggregates. Using the purified enzyme, we showed that HOCl generated by MPO inactivated the enzyme by destroying its prosthetic heme groups and releasing iron. MPO protein was additionally modified by forming high molecular weight aggregates. Before inactivation occurred, MPO chlorinated itself to convert most of its amine groups to dichloramines. When human neutrophils phagocytosed Staphylococcus aureus, they released MPO that was largely inactivated in a process that required production of superoxide. Enzyme inactivation occurred inside neutrophils because it was not blocked when extracellular HOCl was scavenged with methionine. The inactivated enzyme contained a chlorinated tyrosine residue, establishing that it had reacted with HOCl. Our results demonstrate that MPO will substantially inactivate itself during phagocytosis, which may limit oxidant production inside phagosomes. Other neutrophil proteins are also likely to be inactivated. The chloramines formed on neutrophil proteins may contribute to the bactericidal milieu of the phagosome

Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues

We re-determined the near infrared (NIR) spectral signatures (650-980 nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980 nm is cupric CuA, which in the beef heart enzyme has a maximum at 835 nm. The 655 nm band characterises the fully oxidised haem a3/CuBbinuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting 'perturbed' binuclear centre is also characterised by a previously unreported broad 715-920 nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835 nm maximum or the 715 nm band. Our results confirm the ability of NIRS to monitor the CuAcentre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a3/CuBbinuclear centre

The bioenergetic role of dioxygen and the terminal oxidase(s) in cyanobacteria

AbstractOwing to the release of 13 largely or totally sequenced cyanobacterial genomes (see http://www.kazusa.or.jp/cyano and www.jgi.doe.gov/), it is now possible to critically assess and compare the most neglected aspect of cyanobacterial physiology, i.e., cyanobacterial respiration, also on the grounds of pure molecular biology (gene sequences). While there is little doubt that cyanobacteria (blue-green algae) do form the largest, most diversified and in both evolutionary and ecological respects most significant group of (micro)organisms on our earth, and that what renders our blue planet earth to what it is, viz. the O2-containing atmosphere, dates back to the oxygenic photosynthetic activity of primordial cyanobacteria about 3.2×109 years ago, there is still an amazing lack of knowledge on the second half of bioenergetic oxygen metabolism in cyanobacteria, on (aerobic) respiration. Thus, the purpose of this review is threefold: (1) to point out the unprecedented role of the cyanobacteria for maintaining the delicate steady state of our terrestrial biosphere and atmosphere through a major contribution to the poising of oxygenic photosynthesis against aerobic respiration (“the global biological oxygen cycle”); (2) to briefly highlight the membrane-bound electron-transport assemblies of respiration and photosynthesis in the unique two-membrane system of cyanobacteria (comprising cytoplasmic membrane and intracytoplasmic or thylakoid membranes, without obvious anastomoses between them); and (3) to critically compare the (deduced) amino acid sequences of the multitude of hypothetical terminal oxidases in the nine fully sequenced cyanobacterial species plus four additional species where at least the terminal oxidases were sequenced. These will then be compared with sequences of other proton-pumping haem–copper oxidases, with special emphasis on possible mechanisms of electron and proton transfer

Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria

4 pages.-- PMID: 15963511 [PubMed].-- Printed version published on Jul 4, 2005.Plastocyanin and cytochrome c6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b6f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time-resolved analysis showing that both cytochrome c6 and plastocyanin can be efficiently oxidized by the aa3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s−1 for cytochrome c6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.This research was supported by the Spanish Ministry of Education and Science (Grant BMC2003-0458), Andalusian Government (PAI, CVI-0198) and the European Commission through the Human Potential Programme (Contract HPRN-CT-1999-00095). The authors thank Cristina Albarrán for providing the Nostoc cytochrome c6 samples.Peer reviewe